Cocaine abuse is a major medical and public health concern in the United States with more than 2 million chronic, hardcore users and 80,000 cocaine-related ER visits annually. The personal damage and economic harm to society is enormous. Unfortunately, despite extensive work, no clinically useful agent is currently available to effectively treat cocaine addiction. An urgent need exists to develop an effective agent that prevents the behavioral and psychoactive effects of cocaine. One promising alternative approach is to accelerate the breakdown of cocaine to non-harmful products in blood before it reaches the brain by administration of cocaine-metabolizing enzymes. We propose herein to use powerful molecular evolution strategies and high throughput expression and functional screening systems to significantly improve the catalytic activity of cocaine catalysts to achieve the potency required for therapeutic utility. The Specific Aims includes 1) Validate high throughput functional screening platforms using a cocaine analogue;2) In vitro evolution of candidate enzymes for improved cocaine detoxification catalysis by screening highly diversified mutation libraries;and 3) Examine biochemical functions of evolved enzyme. This work will provide the initial research and development effort required to identify cocaine catalytic enzymes through a combinatorial approach of multi-disciplinary research. Since the targeted enzyme has already been developed for other clinical use, we will have the next generation of cocaine catalysts ready to enter preclinical testing and advanced product development if the evolution effort proved successful. Development of the therapeutic agent proposed here will provide significant improvement for the management of cocaine addiction. We propose to use powerful molecular evolution strategies to create cocaine hydrolyzing enzymes that can accelerate the breakdown of cocaine to non-harmful products in blood to prevent neurological and cardiovascular complications. Success of this work will led to the development of novel, safe, and effective medical countermeasures for both emergency detoxification of cocaine overdose, and for chronic reduction of cocaine reinforcing psychoactive effect.